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Significance

The main constituent of the ribosomal stalk is L12, a multicopy protein of the ribosomal 50S subunit that interacts with the translational GTPases in different stages of translation. Using mutagenesis, fast kinetics, and molecular dynamics simulations, we show that charge complementarity between a conserved region on the C-terminal domain of L12 and a conserved region on the G domain of initiation factor 2 (IF2) forms the basis of L12–IF2 interaction, which is crucial for fast association of the ribosomal subunits. Our analysis also suggests that L12 possibly interacts with other G factors by a similar mechanism. This study provides significant mechanistic insights into the interaction of L12 and the translational GTPases at the molecular level.

Abstract

The interaction between the ribosomal-stalk protein L7/12 (L12) and initiation factor 2 (IF2) is essential for rapid subunit association, but the underlying mechanism is unknown. Here, we have characterized the L12–IF2 interaction on Escherichia coli ribosomes using site-directed mutagenesis, fast kinetics, and molecular dynamics (MD) simulations. Fifteen individual point mutations were introduced into the C-terminal domain of L12 (L12-CTD) at helices 4 and 5, which constitute the common interaction site for translational GTPases. In parallel, 15 point mutations were also introduced into IF2 between the G4 and G5 motifs, which we hypothesized as the potential L12 interaction sites. The L12 and IF2 mutants were tested in ribosomal subunit association assay in a stopped-flow instrument. Those amino acids that caused defective subunit association upon substitution were identified as the molecular determinants of L12–IF2 interaction. Further, MD simulations of IF2 docked onto the L12-CTD pinpointed the exact interacting partners—all of which were positively charged on L12 and negatively charged on IF2, connected by salt bridges. Lastly, we tested two pairs of charge-reversed mutants of L12 and IF2, which significantly restored the yield and the rate of formation of the 70S initiation complex. We conclude that complementary charge-based interaction between L12-CTD and IF2 is the key for fast subunit association. Considering the homology of the G domain, similar mechanisms may apply for L12 interactions with other translational GTPases.

Footnotes

Author contributions: S.S. conceived the project idea and designed research; X.G. and C.S.M. performed biochemical experiments; X.G., C.S.M., and S.S. analyzed data; C.L. and J.Å. performed MD simulations; and X.G., C.S.M., C.L., and S.S. wrote the paper.

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